This proposal develops powerful new methods for obtaining efficient site-specific integration at pre-determined genomic sequences. The work elaborates on a successful line of research in my laboratory involving recombination mediated by phage integrases at their compact (30 - 40-bp) attB and attP recognition sites. We have demonstrated that this reaction represents the most efficient site-specific integration system developed to date for higher cells. In the proposed strategy, integration takes place between an attB site present on an incoming DNA vector and a naturally-occurring attP-like sequence located in the mouse genome. The result is covalent integration of the incoming DNA into a pre-specified position in the mouse genome. Directed evolution of phage integrases is used to produce a large library of integration tools of unprecedented efficiency and specificity. With this system, a custom integration tool can be designed for any genomic sequence and used to integrate a DNA insert of large or small size at high efficiency. The DNA to be inserted can be custom designed as desired. Because the method is site-specific, the location of the insertion is automatically known. We develop the appropriate DNA shuffling and genetic screening methods here for generation of a library of custom integration tools. The strategy is scalable to reach essentially all genes in the mouse genome.